Viral entry into permissive cells is the first step in establishing infection and is thus a common and often effective antiviral drug target. However, after replication and assembly of viral particles in the initially infected cell, many viruses suchas HCV can spread to infect additional cells by two routes: cell-free and cell-to-cell spread. Because cell-to-cell spread can be a factor in the establishment of persistent infections and has been implicated in the spread of viral escape mutants during antiviral treatment, elucidating the mechanism(s) of viral cell-to-cell spread provides an opportunity to not only further our understanding of this relatively understudied aspect of virology and pathogenesis, but also perhaps provide insight into global strategies for enhancing the barrier to antiviral resistance during treatment. This is relevant for hepatitis C virus (HCV) because while effective interferon-free direct acting antiviral therapeutic combinations are becoming available for the treatment of HCV, the risk of viral escape has not been determined in less than ideal compliance populations and the astronomical cost of these drugs makes them prohibitively expensive for the majority of the world's HCV-positive populations. To address both this HCV-specific and more general public health issue, our long term goals include elucidating the molecular mechanisms of cell-free and cell-to-cell HCV spread, assessing the role of cell-to-cell spread in the establishment and maintenance of chronic viral infections, and assessing how blocking viral cell- to-cell spread, or not, can impact the synergy potential and barrier to resistance of different combination therapy approaches. The more focused objective of this small pilot R03 proposal is to identify the host cell factors/pathways involved in HCV cell-to-cell spread through directed siRNA knockdown screening interrogating genes already known to be involved in cell-free viral egress from the producer cell or cell-free entry into a target cells. While we already know that cell-free and cell-to-cell entry share some of the same factors and mechanisms (e.g. SRB1, CLDN1, OCLN), we have preliminary data identifying differentially required entry factors. More uniquely, we have also found that certain cellular factors required for the egress of cell-free infectious virus particles, are not required for the cell-to-cell spread. Hence, we hypothesize tha while there are some commonalities between cell-free and cell-to-cell spread, that there key mechanistic differences. Importantly, once identified, these differences can be exploited both as research tools and to develop effective antiviral strategies. As such, this pilot study will provid the fundamental information needed to enable the design of more in-depth mechanistic studies of these critical virus-host interactions and to determine whether factors involved in both routes of spread can serve as highly effective antiviral targets that enhance the barrier to viral resistance, a broad clinically relevant issue for the treatment of viral infections.